Disseminated cytomegalovirus(CMV) infection is a frequent occurrence in AIDS in humans and in SIV-infected rhesus macaques. Reactivation of latent CMV infection is believed to occur as a result of HIV/SIV-induced immunosupression. Since CTL play a major role in control of viral infections, we set out to characterize CMV-specific CTL responses in SIV-infected and uninfected rhesus macaques. Experimental conditions for detection of CMV-specific CTL were initially established in CMV-seropositive, SIV-seronegative rhesus macaques. Autologous fibroblasts infected with a clinical rhesus CMV isolate were used to stimulate freshly isolated peripheral blood mononuclear cells (PBMC). Following in vitro stimulation for two weeks, specific CTL activity against CMV-infected autologous fibroblasts could be detected reproducibly in 4 of 4 CMV-seropositive adult breeder macaques. No CTL activity was detected when PBMC from these animals were stimulated in vitro with uninfected fibroblasts. Similarly, no CMV-specific CTL activity was detected using stimulated PBMC from a CMV-seronegative animal. CMV-specific CTL activity was also detected in two CMV seropositive macaques infected with a live attenuated SIV deficient in nef, LTR and vpr (SIV 3) and in two of 3 macaques chronically infected with a pathogenic SIV strain. The CMV-specific CTL response is MHC class I restricted, as shown by the ability of Brefeldin A, a selective blocker of the class I antigen presentation pathway, to abrogate lysis and by the partial to absent killing of allogeneic targets. In those cases where killing of allogeneic targets was observed, isoelectric focusing of immunoprecipitates of MHC class I molecules showed similar bands, suggesting shared MHC class I alleles. These studies will allow us to prospectively analyze CMV-specific cellular immune responses in SIV-infected animals and examine the host factors that correlate with reactivation of CMV.